Method for characterizing autoimmune disorders

ABSTRACT

The invention relates to methods of characterizing autoimmune diseases by detecting and measuring at least one analyte using multiplexed assay systems. According to one embodiment, a target is detected and measured by different bead sets having different reactants. According to another approach the ratio of self-antigen to autoantibody is measured by exposing a sample suspected of containing self-antigen and autoantibody to a bead set associated with monoclonal antibody specific for the self-antigen and a bead set associated with the self-antigen.

1.0 RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/312,746, filed Aug. 17, 2001, which is incorporated herein byreference.

2.0 FIELD OF THE INVENTION

[0002] The present invention relates generally to methods of, andproducts for, measurement of varied targets in biological fluids. Thepresent invention relates, more specifically, to rapid assays fordiagnosis of autoimmune diseases.

3.0 BACKGROUND OF THE INVENTION

[0003] Analysis of clinical specimens is important in science andmedicine. Multiplexed assays to determine qualitative and/orquantitative characteristics of a specimen are known in the art. Forexample, U.S. Pat. No. 5,981,180 (the “'180 patent”), which is herebyincorporated by reference, discloses methods, instrumentation, andproducts for detecting multiple analytes in a fluid sample by flowcytometric analysis. The products include bead subsets, each bead subsethaving a different reactant bound to the bead. The individual subsetsare prepared so that beads within a subset are relatively homogenous butdiffer in at least one distinguishing characteristic from beads in anyother subset. Therefore, the subset to which a bead belongs can readilybe determined after beads from different subsets are pooled. The methodsinclude pooling the variously labeled subsets prior to assay and mixingthe pooled bead set with a fluid sample to test for analytes reactivewith the various reactants bound to the beads.

[0004] Autoimmunity is a disease condition whereby the body's immunesystem produces autoantibodies against the body's own normal components,i.e. self-antigens, rather than antibodies against foreign substances,i.e. antigens, to the body. The onset of autoimmune disorder isdifficult to diagnose because autoantibodies may be produced from aboutone month prior to as much as thirty years prior to development of sucha disorder. As a result, conducting clinical trials for pharmaceuticalsdirected to preventing autoimmune disorder is problematic; it iseconomically and logistically prohibitive to run trials for an unknownlength of time and up to thirty years in duration.

[0005] There is therefore a need for a method which can better defineclinical presentation of autoimmune disorders. Preferably, such a methodcould enable researchers to identify those patients that would developdisorders in the short term and thus be suitable candidates for clinicaltrials.

4.0 SUMMARY OF THE INVENTION

[0006] The present invention teaches a novel approach to thedifferential diagnosis and/or analysis of many autoimmune diseases.Generally, the invention relates to detecting, in a sample, for exampleof blood, drawn from a patient, the ratio of autoantibody toself-antigen, the presence of self-antigen and variations of theself-antigen, the varied autoantibodies specific for a self-antigen,and/or the relative amount of autoantibody bound to self-antigen.

[0007] In one aspect, the present invention relates to a method ofanalyzing autoimmune disease states, comprising: determining a firstratio of autoantibody to self-antigen in a first sample of blood from apatient by: (a) exposing the blood sample to a pooled population ofsubsets of particles, wherein at least one subset of particles is boundto a reactant capable of binding the autoantibody and at least onesubset of particles is bound to a reactant capable of binding theself-antigen; and (b) detecting the amount of autoantibody and theamount of self-antigen in the blood sample. In some embodiments, thereactant capable of binding the autoantibody is the self-antigen itself,whereas the reactant capable of binding the self-antigen is a monoclonalantibody to the self-antigen. In some embodiments of the invention, theratio is compared against standard ratios representing normal anddiffering degrees of disease states for the disease being analyzed todetermine the presence, absence, onset, or progression of the disease.In some embodiments, the method is repeated over time and the derivedratios are compared one to another to analyze the presence, absence,onset, or progression of the disease.

[0008] In one aspect, the present invention relates to a method ofdetecting a condition indicative of a disease state comprising, (a)providing a pooled population of subsets of particles, wherein theparticles of one subset: (i) are distinguishable from the particles ofanother subset based at least on the fluorescence characteristic of theparticles; and (ii) are associated with a reactant capable of binding ananalyte related to the disease state, wherein the reactant associatedwith one subset of particles is different from the reactant associatedwith another subset of particles; (b) exposing a sample containing atleast one analyte related to the disease state to the pooled populationof subsets of particles to enable the at least one analyte to react witha corresponding reactant; and (c) simultaneously detecting an amount ofanalyte associated with each subset of particles. In some embodiments,the analytes of interest are a self-antigen and its correspondingautoantibody or autoantibodies. In some embodiments, the analytes ofinterest are the variations of the self-antigen and may also be theself-antigen. In some embodiments, the analytes of interest are theautoantibodies corresponding to a self-antigen.

[0009] In one aspect, the present invention relates to a process formeasuring, in a sample, the ratio of an autoantibody to an antigencomprising: (a) exposing the sample to a plurality of assay systems, (b)detecting the amounts of the autoantibody and the antigen, and (c)comparing the amounts of the autoantibody to antigen. A particularsample can have an absence, a presence, or a measurable amount of theautoantibody or of the antigen or both. The sample can be a biologicalfluid or suspension. The biological fluid or suspension can include, butis not limited to, blood, serum, plasma, sweat, tears, urine, sputum,saliva, semen, cerebrospinal fluid, alveolar fluid, lung lavage, gastricfluid, gastric lavage, peritoneal fluid, wound fluid, nasal discharge,bone marrow sample, cyst fluid, or combinations thereof. The biologicalfluid or suspension can be used after dilution with an acceptablediluent, which can be saline.

[0010] In one aspect, the present invention relates to a pooledpopulation of subsets of particles for use in detecting conditionsassociated with autoimmune disease. In some embodiments, the particlesare beads, which are associated with a reactant specific for an analyteof interest and are suitable for use in flow cytometry experiments; thesubsets of particles are distinguishable from one another at least bytheir fluorescence characteristic; and the pooled population comprisesat least two subsets of particles, the first subset of particles beingassociated with a first reactant, which is a monoclonal antibodyspecific for a self-antigen, the second subset of particles beingassociated with a second reactant, which is the self-antigen.

[0011] Specific embodiments of the present invention may be directed toone, some or all of the above-indicated aspects as well as otheraspects, and may encompass one, some or all of the above- andbelow-indicated embodiments as well as other embodiments. Thus, forexample, a method according to the present invention may comprisepredicting the onset of type I diabetes mellitus by repeatedly measuringthe ratio of autoantibody to insulin over time, wherein the ratio ismeasured by using a pooled subset of particles in a flow cytometryexperiment according to, for example, the '180 patent, wherein a firstsubset is a set of beads associated with a monoclonal antibody specificfor insulin and a second subset is a set of beads associated withinsulin.

5.0 DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS 5.1 DEFINITIONS

[0012] The term “particle” refers to a solid phase material, such as amicrosphere or bead, suitable for use in flow cytometric multiplexedassays, for example assays in accordance with the '180 patent.

[0013] The term “subset of particles” refers to a group of particlessharing essentially the same characteristic classification parameters.By “essentially” it is mean that the particles are similar to the extentthat they can be identified as belonging to the same subset of particlesand also distinguished from the particles of another subset. One subsetof particles is distinguishable from another at least based on itsfluorescent characteristic and on the reactant bound to it.

[0014] The term “reactant” refers to a substance associated with themicrosphere or bead, generally bound to the surface of the microsphereor bead, that is capable of coupling with an analyte of interest,suspected of being in the sample to be analyzed. Thus, for example, thereactant might be insulin, which is capable of coupling to itsautoantibody. Or else the reactant might be a monoclonal antibodyspecific for insulin.

[0015] The term “analyte,” “analyte of interest,” “target,” and “targetof interest” are used interchangeably to refer to a substance desired tobe measured and suspected of being present in the sample to be analyzed.Thus, for example, if it is desired to measure the amount of insulin andthe amount insulin autoantibody in a sample to obtain a ratio of insulinto autoantibody, both the insulin and insulin autoantibody are“analytes,” “analytes of interest,” “targets” or “targets of interest.”It is understood that when, for example, a self-antigen such as insulinis the “target,” the particle associated with a reactant specific forthat self-antigen may also bind to a self-antigen which is alreadycomplexed with an autoantibody. Thus, when the target is a self-antigen,it is understood that the target is also the self-antigen/autoantibodycomplex.

[0016] The phrase “variations of a target” refers, for example either tothe cells which produce the analyte, or mutations/variations of theanalyte that can be produced, for example, by dying cells. For example,if insulin is a target, variations of insulin would include the cellwhich produces insulin or altered forms of insulin which may be producedby dying cells.

[0017] The phrase “multiplexed assay” refers to an assay, such as thosedescribed in the '180 patent, capable of making different measurementssimultaneously. “Different measurements” is understood to mean detectionof multiple analytes, or detection of a single analyte by different beadsubsets, or a combination of both. In this context, “simultaneously,” isunderstood to mean that the multiple analytes are detected, or thesingle analyte is detected by different bead sets, or the combination ofmeasurements, is performed in the same assay, for example in the sameflow cytometric run. Typically, a multiplexed assay will be performed ina single vessel containing several sets of particles (i.e. pooledsubsets of particles), such that a single multiplexed assay will providemultiple read-outs of information. Thus, the multiplexed assay of the'180 patent is an example of the application of simultaneous analysis.

5.2 DESCRIPTION

[0018] The present invention relates to the measurement of variedtargets present in the blood of individuals predisposed to or exhibitingvarious forms of a disorder. The inventive methods may be useful in abetter definition of clinical presentation of disorders associated withthe targets measured.

[0019] Disorders which can be characterized by methods according to thepresent invention include autoimmune diseases such as, for example, typeI diabetes mellitus, Grave's disease, psoriasis, Duchenne's musculardystrophy, Hashimoto's thyroiditis, systemic lupus erythematosis,rheumatoid arthritis, seleroderma, Sjögren's syndrome, ulcerativecolitis, Crohn's disease, silicone implant-induced autoimmune reaction,immune deficiency syndrome, hepatitis C, Takayasu's arthritis,phagoneuroglanulomatosis, myasthenia gravis, cirrhosis, Birdshotretinopathy, or anti-coagulant deficiency due to autoantibodies.However, the invention is not limited to characterizing autoimmunedisorders and can be applied to disorders involving circulating antigens

[0020] Generally, the invention is directed to methods forcharacterizing a disorder comprising analyzing a sample comprising atleast one analyte by a multiplexed assay system. In one embodiment, themultiplexed assay system can comprise a pooled population of at leasttwo subsets of particles, wherein the particles of each subset bind thesame analyte but the particles of one subset are associated with areactant specific for the analyte and the particles of a another subsetare associated with a different reactant specific for the same analyte.For example, the particles of one subset may be bound to a firstautoantibody capable of binding a self-antigen and the particles ofanother subset are bound to a second autoantibody capable of binding aself-antigen. A sample containing the self-antigen could then beanalyzed by multiplexed flow cytometric analysis, such as described inthe '180 patent, and results, which may be indicative of the relativeaffinities of the autoantibodies to the self-antigen could providebetter definition of the clinical presentation of the disease.

[0021] In an alternative embodiment, the multiplexed assay comprises atleast two subsets of particles, the particles of a first subset areassociated with a reactant specific for the self-antigen, for example amonoclonal antibody specific for the self-antigen, and the particles ofthe second subset are associated with a reactant specific for theautoantibody, that is the particles are associated with the self-antigenitself. The multiplexed assay could then be use to detect and measureamounts of autoantibody, self-antigen (includingself-antigen/autoantibody complex) in a sample suspected of containingthe autoantibody and self-antigen. The ratio of autoantibody toself-antigen derived from the measurement could provide betterdefinition of the clinical presentation of the disease.

[0022] In an alternative embodiment, the multiplexed assay could performboth assays described above. That is, the multiplexed assay couldcomprise at least at least four subsets of particles, the particles of afirst subset would be associated with self-antigen, the particles of asecond subset would be associated with monoclonal antibody specific forthe self-antigen, the particles of a fourth subset would be associatedwith a first autoantibody to the self-antigen, and the particles offifth subset would be associated with a second autoantibody to theself-antigen. Of course, as many subsets as different autoantibodiescould be used.

[0023] For example, a method according to the present invention could beused to measure the ratio of varied targets present in the blood ofindividuals predisposed to or exhibiting the autoimmune disease Type IDiabetes mellitus. Repeated measurements over time, indicating anincreased amount of autoantibody relative to insulin, could be used topredict onset of diabetes. In an embodiment of the method a pooledpopulation of subsets of particles would be used to measure the ratio ofautoantibody to insulin. Particles of one subset would be associatedwith insulin, which would bind autoantibody in the sample to beanalyzed. Particles of another subset would be associated with amonoclonal antibody to insulin, which would bind insulin andinsulin/autoantibody complex in solution. The ratio so measured can becompared to a standard in which the standard can include a referencevalue for the disease or a previously measured ratio for that samepatient. With respect to the latter, over time, a change in the measuredratio, specifically a higher ratio of autoantibody to insulin, mayindicate depletion of insulin and onset or presence of disease.

[0024] Without being bound by theory, the methods are based on thenotion that blood from individuals predisposed to or exhibiting variousforms of a disorder may present a target in at least two forms that canbe appropriately measured using well-characterized immunoassaytechniques. Autoimmune disorders represent an example of such a concept.Nascent autoantibodies present in an autoimmune disorder should haveseveral populations of antibodies present in the serum. It is postulatedthat for one such population the antibodies are tightly bound to theagent responsible for the development of the autoimmune disorder. It isfurther postulated that also present is another population that may bemore loosely bound to the agent of that may not be bound to any of theagent present in the blood. It is further postulated that the rapidmeasurement of both types of antibody populations will lead to a betterprediction of the clinical condition of the patient. This can beaccomplished by attaching the target of such a disorder or itsassociated antibody to a solid phase for separate assays of the contentsof the blood. As a further extension of this principle, additionalantibodies to a target and/or variations of a target may be added toadditional solid phases for a more complete evaluation of the agentspresent in a sample of blood from a patient.

[0025] The initial observation has been made with insulin autoantibodiesin serum from individuals predisposed to the development of Type IDiabetes mellitus. Type I Diabetes mellitus has been described as anautoimmune disorder. In an experiment performed to demonstrate thisconcept, insulin was coupled to one set of beads and a monoclonalantibody to insulin was coupled to a second set of beads. A mixture ofthese beads (i.e. pooled population of subsets of particles) was thenused in an assay to measure the presence of autoantibodies to insulin.Two distinct populations could be observed in each serum sample. The twopopulations also varied among the individual samples assayed. In some ofthe samples there was exhibited a stronger response to the beads towhich insulin-was coupled. In other samples there was exhibited astronger response to the beads to which the antibody to insulin wascoupled. The pattern was observed in an assay with the mixed set ofbeads matched the pattern observed in assays when each set of beads wasused individually. The observed phenomenon may be indicative of adepletion or loss of circulating insulin sequestered by theautoantibodies. Changes in ratios of unbound to bound insulin mayrepresent a temporal linear progression toward complete manifestation ofdiabetes.

[0026] The phenomenon observed for diabetes should be exhibited in anyand all autoimmune disorders involving circulating antigens and theabove-described methodology is a unique example of a technique that canappropriately measure the components present in an autoimmune disorder.Additional targets, including tumor markers (i.e. tumor antigens),hormones, etc., which may be present in the blood in both a bound andfree form may also benefit from application of methods according to thisinvention. For example, the tumor marker PSA can be found in bound andunbound form, the bound form being PSA bound to a protein that carriesit around the body non-specifically. The disease state, prostate cancer,associated with PSA could therefore be better defined by applying theinventive methodology to analyzing samples suspected of containing PSA.Thus, for example, a sample of blood, drawn from a patient, could beanalyzed by multiplexed flow cytometric analysis using a subset of beadsassociated with PSA and a subset of beads associated with a monoclonalantibody specific to PSA. A ratio of PSA to autoantibody could bederived from those measurements. As with the insulin example above, ifsamples are taken from a patient over time and analyzed a change inratio may be indicative of a change in disease state.

[0027] As described above, additional antibodies to a target and/orvariations of a target may be added to create additional sets of beadsto further enhance the assay for an autoimmune disorder, tumor marker,hormone, etc. The different populations of these targets in a bloodsample can be measured concurrently and temporally, using for examplethe flow cytometric analysis described in the '180 patent, for a morecomplete description of the characteristics of a disorder associatedwith the targets measured. Therefore, the inventive methods can beuseful in a better definition of clinical presentation of disordersassociated with the targets measured.

[0028] Another aspect of the present invention is kits for the detectionor quantitation of an analyte or analytes. The kits can comprise pooledsubsets of particles suitable for characterizing disorders. For example,the kit could include a first and second subset of particles. Theparticles of the first subset are associated with a first reactant, aself-antigen, whereas the particles of the second subset are associatedwith a second reactant, a monoclonal antibody to that self-antigen. Theparticles, apart from the reactant, can be polymeric particles whichrange in size from 0.01 to 1000 micrometers (μm) in diameter. In oneembodiment, the size ranges from 0.1-500 μm. In another embodiment thesize ranges from 1-200 μm. In another embodiment the size ranges from2-12 μm. The particles can be similarly-sized. By “similarly-sized,” itis meant that difference between particles within a set is not more than15%. The particles can be of any shape. In one embodiment, the shape isglobular. However, particles of any other shape can be employed. Theshape of the particle can serve as an additional distinction parameter,which can be discriminated by flow cytometry, e.g., by high-resolutionslit-scanning or by light scatter.

[0029] The kits can include signal ligands for use with sandwich orcompetitive immunoassays. A signal ligand refers to a reactant, which isunassociated to any bead, capable of binding a target and beingdetected. A signal ligand can be, for example, any substance havingassociated therewith a detectable label such as a fluorescently- orradioactively-tagged antibody or antigen. The kit can also contain abinding partner for the signal ligand which forms a complex with forexample, an antibody, antigen, biotin, hapten, or analyte. The kits caninclude sets of particles for use as internal standards. Or else thekits can includes a set or sets of particles for use as controls. Orelse the kits can include sets of particles for use as internalstandards and a set or sets of particles for use as controls.

[0030] A person of ordinary skill will appreciate that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of theinvention.

[0031] For example, although analysis of blood drawn form a patientsuspected of having a disease or being predisposed to a disease, isprincipally described, the multiplexed analysis could be performed onsamples containing controlled amounts of target relating to the diseaseconditions. For example, if a sample containing a controlled amount ofself-antigen is exposed to a pooled subset of particles, wherein theparticles of each of the subsets was associated with a differentreactant, but each of the reactants was specific for the same analyte,for example each subset was associated with a different autoantibody fora self-antigen, then the relative affinity of a self-antigen to varyingautoantibodies may be determined.

[0032] Also, although the illustrated embodiments are particularlyadapted for use with addressable microsphere technology developed byLuminex Corporation, and disclosed for example in U.S. Pat. No.5,981,180, the present invention can be adapted for use with anymultiplexed assay system.

6.0 EXAMPLES

[0033] The following examples are for illustrative purposes only; thescope of the invention is not in any way limited to the particularexamples provided.

6.1 Application of the Methods of the Invention to Diabetes Mellitus,Type I

[0034] Serum samples from individuals predisposed to the development ofType I Diabetes mellitus are tested for insulin autoantibodies by themethods and compositions of the invention. Type I Diabetes mellitus hasbeen described as an autoimmune disorder. In an experiment performed todemonstrate the method of the invention, insulin is coupled to one setof Luminex Beads and a monoclonal antibody to insulin is coupled to asecond set of Luminex Beads. A mixture of these beads is then used in anassay to measure the presence of autoantibodies to insulin by sandwichassays in a fluorescence cytometer. Two distinct populations can beobserved in each serum sample. It is further observed that these twopopulations varied among the individual samples assayed. In some of thesamples a stronger response is exhibited to the beads to which insulinwas coupled. In other samples a stronger response is exhibited to thebeads to which the antibody to insulin was coupled. The pattern observedin an assay with the mixed set of beads matches the pattern observed inassays when each set of beads is used individually.

[0035] The observed phenomenon can be indicative of a depletion or lossof circulating insulin sequestered by the autoantibodies. Changes in theratios of the unbound to bound insulin can represent a temporal linearprogression toward complete manifestation of diabetes.

6.2 Autoantibodies in Diabetes Mellitus, Type I.

[0036] Children with diabetes mellitus, type I, commonly have, in theblood, circulating autoantibodies to glutamate decarboxylase,autoantibodies to the cells of the islets of Langerhans, and/orautoantibodies to insulin. Relatives of such patients have a lowerfrequency of these autoantibodies. The pathogenesis of diabetesmellitus, type I, appears to develop as an activation of autoimmunity inpersons with genetic predisposition and may be influenced byenvironmental factors. An early stage of the pathogenesis is thelymphocytic infiltration of the pancreatic islets of Langerhans,followed by destruction of pancreatic beta cells by cytotoxic Tlymphocytes. A very early and specific response is the development ofimmunoreactivity to glutamate decarboxylase. Anther early response isthe development of immunoreactivity to insulin. Another early responseis the elicitation of antibodies termed IA-2 (insulinoma-associatedprotein-2/protein tyrosine phosphatase).

[0037] Multiple subsets of particles are prepared each with anidentifiable red to orange fluorescence ratio. One subset is conjugatedto insulin by standard methods. A second subset is conjugated toglutamate decarboxylase. A third subset is conjugated to IA-2. Thesubsets are combined and an aliquot combined with a serum sample fromeach of several children at risk for developing diabetes mellitus, typeI. After a short incubation, fluorescein-labeled anti-human (IgG+IgM) isadded and the samples can also be mixed with internal standardsconsisting of known amounts of autoantibodies (to insulin, glutamatedecarboxylase and IA-2) conjugated to particles, to provide absolutevalues for the amount of auto antibodies.

6.3 Analysis of Autoantibodies as a Component of Experimental Diagnosisof Graves'Disease

[0038] Distinct and identifiable subsets particles are prepared: onesubset conjugated to human thyroid microsomal peroxidase (TPO); onesubset to the flavoprotein subunit of succinate dehydrogenase; onesubset conjugated to the extracellular domain of the human TSH receptoror the C-terminal region thereof; and one subset conjugated to tumorsuppressor gene p53. Serum samples of women with familial histories ofGraves'disease and/or Hashimoto's thyroiditis are tested with an aliquotof the pooled particles and a signal molecule in which afluorescein—conjugated anti-human (IgG+IgM) immunoglobulin G is used. Aninternal standard is used to quantify autoantibody levels. Thequantitative results are compared to standard individualradio-immunoassays and a physical diagnosis.

6.4 Multiple Clonal Autoantibodies to the TSH Receptor in Conjunctionwith Graves'Disease

[0039] The method of Example 6.3 is used with the variation that severalsubsets of particles are prepared each comprising a different shortfragment of the C-terminal region of the extra cellular domain of theTSH receptor. The custom oligopeptides of the C-terminal region areobtained from a commercial source (e.g. Bachem) and conjugated to theparticle by any standard method.

We claim:
 1. A method for characterizing autoimmune disease states,comprising: determining a first ratio of autoantibody to self-antigen ina first sample of blood from a patient by: (a) exposing the blood sampleto a pooled population of subsets of particles, wherein at least onesubset of particles is bound to a reactant capable of binding theautoantibody and at least one subset of particles is bound to a reactantcapable of binding the self-antigen; and (b) detecting the amount ofautoantibody and the amount of self-antigen in the blood sample.
 2. Amethod according to claim 1, wherein the reactant capable of binding theautoantibody is the self-antigen, and the reactant capable of bindingthe self-antigen is a monoclonal antibody specific for the self-antigen.3. A method according to claim 2, wherein the self-antigen is insulin.4. A method according to claim 1, further comprising comparing thedetermined ratio to standard ratios representing normal and diseasestates to analyze the presence, absence, onset, or progression of thedisease.
 5. A method according to claim 1, further comprisingdetermining a second ratio of autoantibody to self-antigen in a secondsample of blood from the same patient, wherein the second sample isdrawn subsequently in time to the first sample, and comparing the secondratio to the first ratio to analyze the presence, absence, onset, orprogression of the disease.
 6. A method according to claim 1, furthercomprising determining at least one subsequent ratio of autoantibody toself-antigen in at least one additional sample of blood from the samepatient, wherein each additional sample of blood is drawn subsequentlyin time from the previous sample, and comparing the at least onesubsequent ratio to the first ratio and the second ratio to analyze thepresence, absence, onset, or progression of the disease.
 7. A method ofdetecting a condition indicative of a disease state comprising, (a)providing a pooled population of subsets of particles, wherein theparticles of 10 one subset: (i) are distinguishable from the particlesof another subset based at least on the fluorescence characteristic ofthe particles; and (ii) are associated with a reactant capable ofbinding an analyte related to the disease state, wherein the reactantassociated with one subset of particles is different from a reactantassociated with another subset of particles; (b) exposing a samplecontaining at least one analyte related to the disease state to thepooled population of subsets of particles to enable the at least oneanalyte to react with a corresponding reactant; and (c) simultaneouslydetecting an amount of analyte associated with each subset of particles.8. A method according to claim 7, wherein the at least one analyte is aself-antigen and the reactants are autoantibodies to the self-antigen.9. A method according to claim 7, wherein the sample contains at leasttwo analytes related to the disease state, a first analyte being aself-antigen and a second analyte being an autoantibody to theself-antigen, and wherein a reactant associated with one subset ofparticles is the self-antigen and the reactant associated with anothersubset of particles is the autoantibody.
 10. A method according to claim7, wherein the reactants associated with the subsets of particles arechosen from a self-antigen and variations of the self-antigen.
 11. Amethod according to claim 7, wherein the disease is an autoimmunedisease chosen from type I diabetes mellitus, Grave's disease,psoriasis, Duchenne's muscular dystrophy, Hashimoto's thyroditis,systemic lupus erythematosis, rheumatoid arthritis, scleroderma,Sjogren's syndrome, ulcerative colitis, Crohn's disease, siliconimplant-induced autoimmune reaction, immune deficiency syndrome,hepatitis C, Takayasu's arthritis, phagoneuroglanulomatosis, nyastheniagravis, cirrhosis, Birdshot retinopathy, and anti-coagulant deficiencydue to autoantibodies.